The study of cellular proteins that are targeted by the tumor antigens of papovaviruses is proving increasingly important to our understanding of human cancer, yielding important insights into signal transduction pathways that play a role in cancer. We have identified one of the targets of polyomavirus middle (MT) and small (ST) tumor antigens as protein phosphatase 2A (PP2A), a highly conserved enzyme known to be involved in the control of cell proliferation and apoptosis. This enzyme is important for polyomavirus-induced tumorigenesis and has also has been implicated in human cancer, both as a target of mutation or misregulation and as a potential target for anti-cancer therapies. A long-term goal of the proposed project is to help delineate the molecular mechanisms that regulate PP2A, especially in cell cycle, apoptosis, and cancer. This research will lead to the identification of new cellular drug targets that will allow us to more specifically modulate PP2A function in apoptosis- and cancer-relevant pathways. Drugs that function at the level of these targets would likely be less toxic, and therefore more useful for anti-cancer therapy. Thus, this grant will focus on 1) understanding how MT and ST signal to block apoptosis, 2) understanding how MT and ST alter PP2A function, 3) identifying mechanisms by which PP2A is targeted to specific substrates, and 4) elucidating normal mechanisms of PP2A regulation. In Aim I, we will use wt and mutant STs and MTs and cyclooxygenase-2 inhibitors to determine if MT and ST modulate the PI 3-kinase/Akt/Bad/Bcl-2 anti-apoptotic pathway by PP2A binding and/or cyclooxygenase-2 induction. We will also investigate whether Akt and Bcl-2 are targeted by known PP2A regulatory subunits by using coimmunoprecipitation approaches. In Aim II, we will test which PP2A regulatory subunits are displaced from PP2A in vivo by expression of MT and ST. Moreover, we will investigate normal and MT/ST modulated phosphorylation of a novel family of PP2A targeting subunits. Finally, in Aim III we will investigate the mechanism of regulation of PP2A methylation at the level of the methyltransferase and methylesterase enzymes by testing whether these enzymes are regulated by compartmentalization or phosphorylation.